수용성 파클리탁셀 프로드럭의 합성 및 항암 효능

Abstract

Paclitaxel(Taxol), a natural diterpene isolated from the bark of Taxus brevifolia(Pacific yew), is one of the most effective antitumor agents for the treatment of various human solid tumors such as ovarian carcinoma, breast carcinoma, and melanoma. Unlike other clinical antimitotic agents such as the vinca alkaloids which inhibit the microtubule assembly process, paclitaxel promotes tubulin polymerization and stabilizes the microtubules, therefore cell division is blocked in the late G2 mitotic phase of the cell cycle. Due to limitations of water-solubility, paclitaxel is currently administered in vehicles containing Cremophor EL and 49.7 % ethanol(1:1 v/v). The hypersensitivity reaction occurred by Cremophor EL contained in the formulation when the vehicles are administered intravenously. The symptoms of the hypersensitivity reaction include rapid onset of hypertension, respiratory distress(e. g. bronchospasms), urticaria and rash. In spite of the introduction of the prophylactic use of antihistamines prior to paclitaxel i.v. administration and the extension of the infusion duration time to minimize these allergic reactions, the histamine release effects due to Cremophor EL are not completely eliminated. In order to overcome these problems, preclinical strategy for i.v. administration of paclitaxel has focused on the use of prodrugs in a non-Cremophor EL-containing, as 100 % aqueous as possible, formulation. In this study, a new series of PEGylated paclitaxel prodrugs increased water solubility of paclitaxel was synthesized. Because the efficacy of prodrug is dependent upon the release rate of the active parent drug, various self-immolating spacer groups were introduced between the PEG solubilizing portion and 7-OH of paclitaxel so as to change the rate of enzymatic hydrolysis. Two ways of synthetic route were instituted by the turns of PEGylation to paclitaxel derivatives. PEGylation of 2'-OH protected paclitaxel derivatives with silyl group(method 2) was much more susceptible than that of 2'-OH paclitaxel derivative(method 1) by reason that nucleophilic reaction of mPEG-succinate could be affected by free 2-OH group. This PEGylation susceptibility on method 2 resulted in 78 % yield of prodrug, higher than 60 % on method 1. Physical properties of prodrugs and mPEG-succinate were not different enough to be separated each other by simple purifying method such as recrystallization and extraction, so Prep-HPLC was used to purify the prodrugs. After Prep-HPLC operated in gradient-eluent condition, the prodrugs were successfully purified, especially purity of PP7-5000a was over 99 %. The solubility was defined by the amount of the prodrug compound at which the fluidity occurred and the haze was 0.5 or less than. All prodrugs had water-solubility of 400 mg/mL or more and exhibited improved water-solubility significantly, compared to 0.01 mg/mL or less of paclitaxel. While the PEG-paclitaxel conjugated with simple ester bond was hydrolyzed in rat plasma with a t1/2 of about 7 hr, the rates of hydrolysis for the prodrugs containing various self-immolating spacer group showed considerable variation with t1/2 being from 0.94 min to 42.7 min in rat plasma. It appeared that by introducing the self-immolating spacer groups within conjugates, decreased steric hindrance of paclitaxel in enzymatic breakdown process could lead to rapid rate of enzymatic hydrolysis. In case of non-enzymatic hydrolysis, PP7-5000a showed relatively slow hydrolysis rate in PBS and distilled water with t1/2 of about 26 hr and 200 hr. These slow rates of non-enzymatic hydrolysis can guarantee the stability of the prodrug against the problem of paclitaxel precipitation caused by hydrolysis. To evaluate the antimetastaic potential of paclitaxel and PP7-5000a, the effect of the drugs on the development of melanoma lung colonies in C57B/6 mice following i.v. administration of metastatic murine B16/F10 melanoma cells was estimated. Paclitaxel and the prodrug were administered intravenously, once daily for 10 days. Compared to vehicle, % reductions of melanoma lung colonies were 46.9 in the dose of 5 mg/kg paclitaxel, 24.5 in the dose of 5 mg/kg prodrug(0.71 mg paclitaxel equivalent/kg), and 40.0 in the dose of 10 mg/mL prodrug(1.42 mg paclitaxel equivalent/kg). It might be considered that the efficacy of the prodrug, in equivalent dose of paclitaxel, is higher than that of paclitaxel. The enhanced efficacy of paclitaxel by the prodrug might have resulted from the longer circulating lifetime of paclitaxel and the specific affinity of the prodrug for tumor cells.